Growth factors, such as fibroblast growth factor (FGF), are ligands for receptor tyrosine kinases (RTK) and are involved in stimulating cellular proliferation and migration. As a result, growth factor and RTK antagonists may be involved in conditions involving uncontrolled growth and vascularization.
Sprouty was initially identified as an antagonist of FGF signaling in the developmental pathway of Drosophila tracheal branching morphogenesis. An FGF family member, Branchless, is the critical determinant of tracheal branching pattern through inducing cell migration which results in branching morphogenesis. It was determined that Branchless induces expression of a potent negative signal, Sprouty, that inhibits branching morphogenesis and was found to act as an negative regulator of tracheal development. Loss of function mutations in the Sprouty gene led to increased tracheal branching while gain of function mutations severely blocked tracheal development. Overexpression of Branchless overrode the Sprouty loss of function mutation indicating that Sprouty""s antagonist activity was specific for the FGF activity in this developmental pathway (Hacohen et al. (1998), Cell, 92: 253-263; Metzger et al. (1999), Science, 284:1635-1639).
Currently, there are three known human homologs of the Drosophila Sprouty protein denoted human Sprouty-1, Sprouty-2 and Sprouty-3 (genes denoted h-Spryl, h-Spry2, h-Spry3). H-Spry-2 encodes an approximately 35 Kd protein which contains a cysteine-rich domain. This domain is highly conserved among the human homologs (Hacohen et al. (1998), Cell, 92: 253-263).
The Sprouty mouse homolog (sprouty-2, encoded by gene mSpry-2) also was found to regulate bronchial branching patterns in the developing lung. In this system, mSpry-2 inhibited FGF10-induced bronchial branching. The mSpry-2 amino acid sequence is 97% homologous to h-Spry2, indicating that this protein may be highly conserved among different species (Tefft et al. (1999), Curr Biol, 9: 219-222). Another mouse homolog, sprouty-4, has also been identified (De Maximy et al. (1999), Mech. Dev. 81, 213-216).
Other evidence indicates that Sprouty may act as a general inhibitor of RTK activity. In addition to its inhibitory effects on FGF signaling, Sprouty expression was shown to antagonize epidermal growth factor (EGF) action in the developing Drosophila eye imaginal disc, larval peripheral nervous system, embryonic central nervous system, developing wing and developing ovary (Casci et al. (1999), Cell, 96: 655-665, Kramer et al. (1999), Development, 126: 2515-2525). Sprouty also inhibits the action of an unrelated RRX, Torso, which initiates the development of terminal structures in the developing Drosophila embryo (Casci et al. (1999), Cell, 96: 655-665).
Sprouty is an intracellular protein which associates with the plasma membrane through its cysteine-rich domain. Sprouty acts within the mitogen activated protein (MAP) kinase signaling cascade, and its inhibitory action has been mapped to be downsteam of the RTK and upstream of ras. This suggests that Sprouty may be binding to adaptor proteins, such as Drk (the Drosophila homolog of Grb2) and Gap1, within the MAP kinase signaling cascade (Casci et al. (1999), Cell, 96: 655-665).
The autonomous manner in which Sprouty works in the eye is different from the non-autonomous manner in which it acts in the tracheal system. It has been speculated that Sprouty may not only act intracellularly to repress signal transduction but may also act on a second signaling relay to repress cell fate in the neighbors of Sprouty-expressing cells (Placzek et al. (1999), Current Biol., 9: R506-510).
Many growth factors, such as FGF, EGF, platelet-derived growth factor and insulin-like growth factor, elicit their response through RTKs. Activation of the RTK initiates signaling through the MAP kinase cascade. As a result, the MAP kinase signaling pathway is involved in many cellular actions including proliferation, migration, angiogenesis, and organogenesis.
Thus, there is a need for growth factor and MAP kinase signaling antagonists, such as Sprouty family members, which may be useful in modulating cell growth, migration and vascularization.
The compositions of the present invention include novel isolated polypeptides, in particular, novel human growth factor antagonist proteins and active variants thereof isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
A nucleotide sequence encoding a growth factor antagonist protein designated FGFAn-Hy is set forth in SEQ ID NO: 1, and its deduced amino acid sequence is set forth in SEQ ID NO: 2. This growth factor antagonist protein is believed to play a role in the mitogen activated protein (MAP) kinase signaling pathway involving receptor tyrosine kinases (RTKs) and ras. The polypeptide set out in SEQ ID NO: 2 is 300 amino acids in length, and appears to have no readily identifiable signal sequence, indicating that it is most likely an intracellular protein like the Drosophila growth factor antagonist protein Sprouty. The polypeptide of SEQ ID NO: 2 displays amino acid homology with the Drosophila Sprouty, as well as with mammalian family members human Sprouty-1, -2 and -3 and mouse Sprouty-2 and -4. An alignment of FGFAn-Hy and other members of the sprouty family is shown in FIG. 1. FGFAn-Hy is most closely related to mouse sprouty-4 (92% amino acid sequence identity). Additional family members can be identified using SEQ ID NO: 1 as a molecular probe.
The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a portion thereof corresponding to the full length or mature protein. The isolated polynucleotides of the invention further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; a polynucleotide comprising the full length protein coding sequence of SEQ ID NO: 1; and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NO: 1. The polynucleotides of the present invention also include, but are not limited to, polynucleotides that encode polypeptides with growth factor antagonist activity and that hybridize under stringent hybridization conditions to the complement of (a) the nucleotide sequence of SEQ ID NO: 1, or (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homolog of any of the proteins recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide having an amino acid sequence of SEQ ID NO: 2. The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a portion thereof corresponding to the full length or mature protein. Polypeptides of the invention also include polypeptides with growth factor antagonist activity that are encoded by (a) polynucleotides set out in SEQ ID NO: 1; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of the growth factor antagonist protein sequence of SEQ D NO: 2 and xe2x80x9csubstantial equivalentsxe2x80x9d thereof (e.g., with 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that retain growth factor antagonist activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.
Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The invention also relates to methods for producing polypeptides of the invention comprising growing a culture of the cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein from the cells or the culture medium in which the cells are grown. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect or quantify the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Voltrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
Where the polypeptide has growth factor antagonist or MAP kinase signaling antagonist activity, the polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for the prevention and/or treatment of disorders involving cell growth and proliferation, such as angiogenesis, or any of the disorders described below. Where the polypeptide promotes cell growth and proliferation, polypeptides and polynucleotides can be utilized to promote tissue growth, for example, as as part of treatment for increasing vascularization, wound healing, or any of the other disorders described herein.
The methods of the present invention further relate to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions. The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited herein.
Mutations in the FGFAn-Hy gene may be associated with uncontrolled cellular proliferation, such as cancer, and the identification of such mutations and detection of these mutations in subjects may provide important diagnostic and prognostic information.
The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.
The methods of the invention also include methods for the treatment of disorders as recited above which may involve the administration of such compounds to individuals exhibiting symptoms or tendencies related to disorders as recited herein. In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising the step of administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene expression or target protein activity.